Calcium influx through neuronal voltage-sensitive calcium channels (VSCC S) mediates nociceptive information in the spinal dorsal horn. In fact, spinally administered VSCC S blockers, such as omega-conotoxin MVIIA, have analgesic effect apart of their low therapeutic index and many side effects. Here we study the analgesic potential of Ph alpha 1beta, a calcium channel blocker, in rodent models of acute and persistent pain. Spinally administered Ph alpha 1beta showed higher efficacy and long-lasting analgesia in a thermal model of pain, when compared with omega-conotoxin MVIIA. Moreover, Ph alpha 1beta was more effective and potent than omega-conotoxin MVIIA not only to prevent, but especially to reverse, previously installed persistent chemical and neuropathic pain. Furthermore, the analgesic action of both toxins are related with the inhibition of Ca2+-evoked release of pro-nociceptive neurotransmitter, glutamate, from rat spinal cord synaptosomes and decrease of glutamate overflow in cerebrospinal fluid. When side effects were assessed, we found that Ph alpha 1beta had a therapeutic index wider than omega- conotoxin MVIIA. Finally, recombinant Ph alpha 1beta expressed in Escherichia coli showed marked analgesic activity similar to the native toxin. Taken together, the present study demonstrates that native and recombinant Ph alpha 1beta have analgesic effects in rodent models of pain, suggesting that this toxin may have potential to be used as a drug in the control of persistent pathological pain.

BACKGROUND AND PURPOSEPeptides from venomous animals have long been important for understanding pain mechanisms and for the discovery of pain treatments. Here, we hypothesized that Phα1β, a peptide from the venom of the armed spider Phoneutria nigriventer, produces analgesia by blocking the TRPA1 channel. EXPERIMENTAL APPROACHCultured rat dorsal root ganglion (DRG) neurons, human fetal lung fibroblasts (IMR90) or HEK293 cells expressing the human TRPA1 (hTRPA1-HEK293), human TRPV1 (hTRPV1-HEK293) or human TRPV4 channels (hTRPV4-HEK293), were used for calcium imaging and electrophysiology. Nociceptive responses induced by TRPA1, TRPV1 or TRPV4 agonists or by bortezomib were investigated in mice. KEY RESULTSPhα1β selectively inhibited calcium responses and currents evoked by the TRPA1 agonist, allyl isothiocyanate (AITC), on hTRPA1-HEK293, IMR90 fibroblasts and DRG neurons. Phα1β did not affect calcium responses evoked by selective TRPV1 (capsaicin) or TRPV4 (GSK 1016790A) agonists on the various cell types. Intrathecal (i.t.) and intraplantar (i.pl.) administration of low doses of Phα1β (up to 300 pmol per paw) attenuated acute nociception and mechanical and cold hyperalgesia evoked by AITC (i.t. or i.pl.), without affecting responses produced by capsaicin or hypotonic solution. Notably, Phα1β abated the TRPA1-dependent neuropathic pain-like responses induced by bortezomib. In vitro and in vivo inhibition of TRPA1 by Phα1β was reproduced by a recombinant form of the peptide, CTK 01512-2. CONCLUSIONS AND IMPLICATIONSPhα1β and CTK 01512-2 selectively target TRPA1, but not other TRP channels. This specific action underlines the potential of Phα1β and CTK 01512-2 for pain treatment.Abbreviations AITC, allyl isothiocyanate; BTZ, bortezomib; CIPN, chemotherapy-induced peripheral neuropathy; DRG, dorsal root ganglia; i.pl., intraplantar; IMR90, human fetal lung fibroblasts; PWT, paw withdrawal threshold; VGCCs, voltage-gated calcium channels

The treatment with the chemotherapeutic agent paclitaxel produces a painful peripheral neuropathy, and is associated with an acute pain syndrome in a clinically significant number of patients. However, no standard therapy has been established to manage the acute pain or the chronic neuropathic pain related to paclitaxel. In the present study, we evaluated the analgesic potential of two N-type voltage-gated calcium channel (VGCC) blockers, ω-conotoxin MVIIA and Phα1β, on acute and chronic pain induced by paclitaxel. Adult male rats were treated with four intraperitoneal injections of paclitaxel (1+1+1+1mg/kg, in alternate days) and the development of mechanical hyperalgesia was evaluated 24h (acute painful stage) or 15days (chronic painful stage) after the first paclitaxel injection. Not all animals showed mechanical hyperalgesia 24h after the first paclitaxel injection, but those that showed developed a more intense mechanical hyperalgesia at the chronic painful stage. Intrathecal administration (i.t.) of ω-conotoxin MVIIA (3-300pmol/site) or Phα1β (10-300pmol/site) reduced the mechanical hyperalgesia either at the acute or at the chronic painful stage induced by paclitaxel. When administered at the acute painful stage, ω-conotoxin MVIIA (300pmol/site, i.t.) and Phα1β (300pmol/site, i.t.) prevented the worsening of chronic mechanical hyperalgesia. Furthermore, Phα1β (30-300pmol/site, i.t.) elicited less adverse effects than ω-conotoxin MVIIA (10-300 pmol/site, i.t.). Taken together, our data evidence the involvement of N-type VGCC in pain sensitization induced by paclitaxel and point out the potential of Phα1β as a safer alternative than ω-conotoxin MVIIA to treat the pain related to paclitaxel.

The marine snail peptide ziconotide (x-conotoxin MVIIA) is used as an analgesic in cancer patients refractory to opioids, but may induce severe adverse effects. Animal venoms represent a rich source of novel drugs, so we investigated the analgesic effects and the side-effects of spider peptide Pha1b in a model of cancer pain in mice with or without tolerance to morphine analgesia. Cancer pain was induced by the inoculation of melanoma B16-F10 cells into the hind paw of C57BL ⁄ 6 mice. After 14 days, painful hypersensitivity was detected and Pha1b or x-conotoxin MVIIA (10-100 pmol ⁄ site) was intrathecally injected to evaluate the development of antinociception and side-effects in control and morphine-tolerant mice. The treatment with Pha1b or x-conotoxin MVIIA fully reversed cancer-related painful hypersensitivity, with long-lasting results, at effective doses 50% of 48 (32-72) or 33 (21-53) pmol ⁄ site, respectively. Pha1b produced only mild adverse effects, whereas x-conotoxin MVIIA induced dose-related side-effects in mice at analgesic doses (estimated toxic dose 50% of 30 pmol ⁄ site). In addition, we observed that Pha1b was capable of controlling cancer-related pain even in mice tolerant to morphine antinociception (100% of inhibition) and was able to partially restore morphine analgesia in such animals (56 AE 5% of inhibition). In this study, Pha1b was as efficacious as x-conotoxin MVIIA in inducing analgesia in a model of cancer pain without producing severe adverse effects or losing efficacy in opioid-tolerant mice, indicating that Pha1b has a good profile for the treatment of cancer pain in patients. (Cancer Sci 2013; 104: 1226-1230

Phα1β is a potent toxin obtained from the spider Phoneutria nigriventer that blocks neuronal voltage-sensitive Ca(2+) channels. This study compared the antiallodynic effects of Phα1β, ω-conotoxin MVIIA and morphine in mice and their side effects in rats. Mechanical allodynia was measured in mice receiving single intrathecal administration of Phα1β, ω-conotoxin MVIIA or morphine before or after the incisional plantar procedure. The effect of the treatments on cardiovascular profile and global neurological were evaluated in rats. The expression of pro or anti-inflammatory cytokines of human polymorph mononuclear cells was also evaluated. Preemptive use of ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site) induced shorter antiallodynic effect than Phα1β (100 pmol/site) in mice. Post-incision administration of Phα1β (200 pmol/site) induced longer mechanical antiallodynic effect than ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site). Intrathecal injection of Phα1β (200 pmol/site) and morphine (433 pmol/site) did not change while ω-conotoxin MVIIA (100 pmol/site) increased the heart rate in rats 3 h after its administration. Phα1β (200 pmol/site), ω-conotoxin MVIIA (100 pmol/site) and morphine (433 pmol/site) did not change mean arterial pressure 0.5 and 3 h after their administration. The treatments did not alter neurological performance assessed by global neurological evaluation and open-field test. The tested drugs did not induced expression of pro or anti-inflammatory cytokines in CD4 monocytes. In conclusion, preemptive administration Phα1β in mice induced longer antiallodynic effect than ω-conotoxin MVIIA and morphine. Phα1β also induced a longer mechanical antiallodynic effect than ω-conotoxin MVIIA and morphine when used after the surgical incision. The present results suggest that Phα1β has a potential application in the management of postoperative pain with low side effects.